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Persistently infective sendai virus vector

a virus and persistent infection technology, applied in the field of virus vectors, can solve the problems of low species specificity, high gene expression activity of virus, and inability to understand the mechanism of persistent infection, so as to achieve the effect of reducing cytotoxicity and maintaining expression

Active Publication Date: 2010-08-05
NAT INST OF ADVANCED IND SCI & TECH
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Benefits of technology

[0051]The present invention provides a non-transmissible, recombinant Sendai virus vector persistently expressing an exogenous gene. That in turn achieves a high-safety genetic information expression system which can exist independently from chromosomes, which was impossible with conventional gene therapy vectors. The vector is useful in introducing gene therapy drugs. In addition, the techniques used to produce the recombinant virus vector of the present invention is applicable to various virus vectors by changing the gene sequence in a marker gene (e.g. a drug-resistant gene) or vector viral genes. Furthermore, the persistent infection-related mutation (the mutation of the amino acid at position-1618 in L protein) described in the present invention provides a potentially useful genetic material because presumably by introducing and substituting the recombinant Sendai virus vector for another recombinant vector or virus that is not persistently infective, the latter's cytotoxicity is attenuated and its expression becomes sustainable.

Problems solved by technology

The approach has been so far not successful.
Furthermore, the virus exhibits high gene expression activity and low species-specificity.
Nevertheless, the mechanism of persistent infection is not well understood.

Method used

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  • Persistently infective sendai virus vector

Examples

Experimental program
Comparison scheme
Effect test

example 1

Producing Recombinant Sendai Virus (rNa151MFL and rNa151MF)

(1) Producing Recombinant Sendai Virus Producing Vector

[0157]A cDNA obtained by cloning the full length cDNA of Sendai virus strain C1.151 or Nagoya strain was separated into three fragments. A T7 promoter sequence and a guanidine residue including three bases were inserted in this order immediately upstream of the sequence containing a Sendai virus cDNA in the sequence of a fragment containing SeV: 1-2875 (pBSK / Na(3′-E) [derived from Nagoya strain], pBSK / 151(3′-E) [derived from strain C1.151]) (pBSK / Na(3′+X+3G), pBSK / 151(3′+X+3G)). A SeV: 15351-15384 part was cut out from a fragment containing SeV: 10479-15384 (pBSK / Na(E-5′), pBSK / 151(E-5′)). That part, immediately downstream of which the hairpin ribozyme sequence of a tobacco ring spot virus and a T7 RNA polymerase termination sequence were inserted in this order, was recloned into pET30a(+) (Novagen) (pET / Na(5′+HrD), pET / 151(5′+HrD)). Furthermore, this fragment of SeV: 15...

example 2

Confirming Persistent Infectiveness of Recombinant Sendai Virus in Cells

[0161]CV-1 cells or LLCMK2 cells were plated on a 12-well plate and cultured for 24 hours. After the culturing, the virus suspensions were diluted with a medium to M.O.I.=100 and distributed in the wells to cause infection at 37° C. The cells were washed 24 hours later. Then, a medium containing no virus was added. The infected cells were observed to see whether they were dead or alive while culturing them at 37° C. In addition, infection of the cells was confirmed by a fluorescent antibody test using an antibody against Sendai virus.

[0162]As shown in FIG. 2, cytotoxicity was observed in the CV-1 cells infected with rNa151MF about as late as 4 days after the infection. No cytotoxicity could be observed in CV-1 cells infected with rNa151MFL, similarly to CV-1 cells infected with strain C1.151. Later, 10 days after the infection, marked omission of the virus from the infected cells was confirmed in the CV-1 cells ...

example 3

Producing Exogenous-Gene-Inserted rNa151MFL Strain (1); Integrating Exogenous Gene Insertion Site

[0164]A Not I recognition sequence was inserted to pBSK / Na(3′+X+3G) after SeV: 119 by Quikchange Site-directed Mutagenesis II (STRATAGENE) by using 5′-GCCAAAGTTCACGCGGCCGCAGATCTTCACGATGGCCGGGTT GT-3′ (SEQ ID NO 11 (sense strand) in the Sequence Listing) and 5′-ACAACCCGGCCATCGTGAAGATCTGCGGCCGCGTGAACTTTG GC-3′ (SEQ ID NO 12 (antisense strand) in the Sequence Listing) as an exogenous-gene-insertion-site producing primer (pBSK / Na(3′+Not)).

(2) Introducing Exogenous Gene (EGFP Gene)

[0165]An EGFP gene was amplified from pEGFP-C1 (Clontech) by using two primers, 5′-ACTTGCGGCCGCTCGCCACCATGGTGAGCAAGGGCGAGGA-3′ (SEQ ID NO 13 (N terminus end) in the Sequence Listing) and 5′-ACTTGCGGCCGCGATGAACTTTCACCCTAAGTTTTTCTTAGAC GGCCGCTTTACTTGTACAGCTCGTCCA-3′ (SEQ ID NO 14 (C terminus end) in the Sequence Listing) as EGFP gene insertion primers. The termini of the obtained double-stranded DNA were cleaved with ...

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Abstract

A persistently infective virus vector is produced by using a gene so modified as to encode an amino acid sequence including a valine substituted for an amino acid residue at position-1618 in the amino acid sequence for an L protein of a persistently non-infective Sendai virus. A non-transmissible, persistently infective virus vector is also produced by defecting or deleting at least one of M gene, F gene, and HN gene. These virus vectors have no cytotoxicity, can achieve the sustained gene expression over a long period of time, is safe, and is therefore useful.

Description

TECHNICAL FIELD[0001]The present invention relates to virus vectors which is useful for gene therapy among other purposes, stably expresses exogenous genes over a long period of time, or is furthermore non-transmissible and safe. The invention also relates to genetic material for assembling the vector.BACKGROUND ART[0002]In gene therapy for inherited metabolic disorder and other diseases, an introduced exogenous gene is expected to be continuously expressed over a long period of time. This objective has been achieved conventionally by incorporating genetic information into a chromosome of a host by means of a retrovirus vector. Safety is a concern, however, because there have been some clinical reports of cells turning cancerous under the influence of the integrated genes. A suggested approach to the problem is to develop a genetic information expression system which is able to exist stably and independently from chromosomes. The approach has been so far not successful.[0003]Sendai ...

Claims

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Application Information

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IPC IPC(8): C12N7/04C07H21/04C12N15/63A61K31/711A61K35/76A61K48/00A61P35/00C12N1/15C12N1/19C12N1/21C12N5/10C12N7/00C12N7/01C12N7/02C12N15/09C12N15/86C12P21/02
CPCA61K35/12A61K35/13A61K38/162C12N2760/18843C12N15/86C12N2760/18821C12N7/00A61P35/00
Inventor NISHIMURA, KENSEGAWA, HIROAKINAKANISHI, MAHITO
Owner NAT INST OF ADVANCED IND SCI & TECH
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